In addition, this work implies that Wnt5a shifts DCs from glycolysis towards FAO in the melanoma microenvironment and this metabolic program effectively inhibits effector T cell activation while driving Treg cell differentiation. The Wnt5a–catenin signaling Pathway Regulates DC Fatty Acid Oxidation via the PPAR–CPT1A Axis Previous investigators have proposed that activation of AMP-activated protein kinase (AMPK) by the AMP analog, AICAR, would antagonize the glycolytic surge required for DC maturation (Krawczyk et al., 2010). evasion and immunotherapy resistance. expression ((Figures 1G, H, S1K). Together, these data reveal that melanoma tissues shift the metabolism of local DC populations from a glycolytic state toward OXPHOS in a Wnt5a-dependent manner. Open in a separate window Figure 1 Melanoma-derived Wnt5a Alters DC Energy MetabolismA. Lactate in BMDC culture media from 0C48 hours with rWnt5a treatment. n=6. B. Qrt-PCR analysis of and expression in DCs following rWnt5a treatment. n=3. C. ECAR (milli-pH units/minute, normalized to 0 min) of untreated (UT) vs. rWnt5a pretreated DCs. Arrow indicates LPS injection. n=6. D. OCR (pico-moles/minute) of DCs pre-treated with rWnt5a or rWnt3a. n=6. Oligo, oligomycin. FCCP, uncoupling agent. Rot, rotenone. E. ECAR of DCs pre-treated with rWnt5a or rWnt3a. n=6. 2DG, 2-deoxyglucose. F. OCR of DCs injected with media alone or concentrated conditioned media (CM) from and (Holtzhausen et al., 2015). Altogether, these data indicate that inhibition of DC glycolysis and inhibition of DC OXPHOS would have reciprocal effects on Treg cell development. Indeed, co-culturing 2-DG-treated or Wnt5a-treated DCs with na?ve CD4+ T cells generated enhanced Treg cell differentiation while inhibition of DC OXPHOS with oligomycin (oligo) eliminated these Treg cell populations (Figures 2B). Together, these findings imply that Wnt5a drives Treg cell differentiation in the melanoma microenvironment by promoting DC OXPHOS. This is consistent with previous data showing that Wnt3a neither regulates DC metabolism nor promotes DC-mediated Treg cell generation (Figures 1D, E) (Holtzhausen et al., 2015). To examine this question more directly, we purified tumor-infiltrating DCs from (Figure 2C). In summary, metabolic reprogramming plays a central role in Wnt5a regulation of DC functionality and determines whether a DC drives effector T cell expansion versus Treg cell differentiation (Figure 2D). Open in a separate window Figure 2 DC Function is Regulated by Cellular MetabolismA. T cell proliferation assay: DCs loaded with OVA257-264 peptide, pre-treated with rWnt5a or LH-RH, human 2DG, stimulated with LPS, and co-incubated with OT-I splenocytes. CD3+CD8+ T cell proliferation measured by CellTrace Violet (CTV) LH-RH, human dilution. n=3. Representative flow cytometry CTV dilution assay based on 3 independent experiments. Gated on CD3+CD8+ T cells. B. DCs treated with Wnt5a, 2-DG, or Oligo prior to Treg cell assay measuring DC-induced CD4+FoxP3+ Treg cells. n=3. Representative flow cytometry plot of Treg cell analysis based on 3 independent experiments. C. Treg cell analysis of inguinal lymph nodes by flow cytometry. Representative of 3 independent experiments. 4 mice/group. D. Schematic illustrating the dynamic spectrum of DC-induced T cell responses based on their metabolic alteration. UT untreated. KD, knockdown. All data are mean +/? SEM. Treg cell assay measuring DC-induced CD4+FoxP3+ Treg cells. n=3. Treg cell assay measuring DC-induced CD4+FoxP3+ Treg cells following treatment with either rWnt5a or rWnt5a+ETO. n=4/group. LH-RH, human and following the adoptive transfer of conditioned DCs into expression in the DC2.4 myeloid DC line and determined the ability of the resulting DC2.4-CPT1A-silenced cell line to induce Treg cell differentiation as well as to promote effector T cell proliferation relative to the DC2.4-NTC control cell line (Figure S4). This revealed that genetically targeting CPT1A in the DC2.4 line effectively made these DCs resistant to PROK1 Wnt5a-induced Treg cell development while promoting their ability to stimulate CD8+ T cell proliferation (Figures LH-RH, human 3H, I). To demonstrate that genetic silencing of CPT1A can have similar effects in primary DCs, we engineered a CPT1A-specific shRNA-expressing lentiviral vector and LH-RH, human transduced BMDCs before performing OT-I CD8+ T cell proliferation assays (Figures S3D, E). These experiments indeed demonstrated primary CPT1A-silenced DCs induce potent CD8+ T cell proliferation while maintaining resistance to Wnt5a-induced tolerization (Figure 3J). Overall, these data provide a.